Non-segmented negative strand (NNS) RNA viruses, such as rabies virus (RABV), human respiratory syncytial virus (HRSV), human parainfluenza virus type 3 (HPIV3), and Ebola virus (EBOV), pose continuing threats to human health, but there are currently no established prophylactic and/or therapeutic countermeasures against most NNS RNA viral diseases. NNS RNA viruses possess a multifunctional RNA-dependent RNA polymerase (RdRp) large (L) protein, which catalyzes all enzymatic reactions required for viral RNA biogenesis (e.g., RNA synthesis, 5?-capping, cap methylation at the guanine-N7- and ribose-2?-O-positions, 3?-polyadenylation). All these enzymatic activities of the L proteins are unique and potentially druggable. Our goals are to elucidate the molecular mechanisms of RNA synthesis and processing with the L proteins and to develop anti-viral agents against them. To dissect the roles of the L proteins in RNA biosynthesis, we have developed a number of in vitro rhabdoviral RNA synthesis and processing systems for prototypic vesicular stomatitis virus (VSV) and RABV. Using these systems, we discovered that rhabdoviral L proteins catalyze unconventional mRNA capping with a novel GDP polyribonucleotidyltransferase (PRNTase, EC 2.7.7.88) domain. However, the mechanisms of pre- mRNA capping and methylation coupled to mRNA chain elongation remain largely unknown. Furthermore, virus- specific functions of L proteins have not been studied. We hypothesize that L proteins catalyze common enzymatic reactions via evolutionary conserved elements, but manifest virus-specific functions via diversified elements. This hypothesis will be rigorously tested by the following specific aims: to elucidate the mechanisms of (1) co-transcriptional mRNA maturation by rhabdoviral L proteins, (2) cap formation by pneumoviral and paramyxoviral L proteins, and (3) transcription and replication by filoviral L proteins. In Aim 1, we will determine the timing and order of pre-mRNA processing by the VSV L protein during mRNA chain elongation, leading to a new model of co-transcriptional mRNA maturation by L proteins of rhabdoviruses and, by extension, other NNS RNA viruses. In Aim 2, we will reveal common and diversified functions of putative PRNTase and methyltransferase domains of the HRSV and HPIV3 L proteins in mRNA cap formation. In Aim 3, we will provide a novel model for genome replication by the EBOV L protein that is significantly different from that by other NNS RNA viral L proteins. Collectively, the proposed studies will open up a new frontier in understanding how diversified NNS L proteins carry out each step of RNA synthesis and processing together with their cognate co- factor proteins. Our studies will provide foundations for the future development of antiviral agents targeting unique L domains of these significant NNS RNA viruses.